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Packaging cells for recombinant adenovirusRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal Cell, The Polynucleotide Is Encapsidated Within A Virus Or Viral CoatPackaging cells for recombinant adenovirus description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060183232, Packaging cells for recombinant adenovirus. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of PCT International Patent Application No. PCT/EP2004/052428, filed on Oct. 4, 2004, designating the United States of America, and published, in English, as PCT International Publication No. WO 2005/033320 A1 on Apr. 14, 2005, which claims priority to PCT International Patent Application No. PCT/EP03/50679, filed on Oct. 2, 2003, the contents of the entirety of each of which are hereby incorporated herein by this reference. TECHNICAL FIELD [0002] The invention relates to the field of biotechnology and molecular and cell biology; more in particular, to packaging cells and use thereof for the generation of batches of recombinant adenovirus. BACKGROUND [0003] Replication-deficient recombinant adenoviruses are useful, for instance, in gene therapy and for vaccination purposes. They usually lack the E1-region of an adenovirus and are, therefore, propagated on complementing cells providing the E1 sequences. The packaging cells provide all information required for replication of a vector that is packaged in the cells to form recombinant viral particles. An example of a packaging cell is a 293 cell, which contains nucleotides 1-4344 of the adenovirus 5 genome (Louis et al., 1997), which include the 5' inverted terminal repeat (ITR), the packaging signal, the E1A and E1B coding sequences and the pIX coding sequences. [0004] Overlap between the adenoviral sequences in the packaging cell and the vector may lead to homologous recombination between these sequences, resulting in the generation of replication-competent adenovirus (RCA) (Lochmuller et al., 1994). This problem has been solved by using a packaging system consisting of a cell line and a vector that are matched to each other by lacking such overlapping sequences (Fallaux et al., 1998; U.S. Pat. No. 5,994,128). One example of a particularly useful cell line in such applications is the PER.C6.RTM. cell line (U.S. Pat. No. 5,994,128; Nichols et al., 2002). [0005] Recently, it was reported that upon use of PER.C6.RTM. cells in conjunction with a vector that still contained 177 bp sequence homology with the E1 sequences in the genome of PER.C6.RTM., a single cross-over event could result in the generation of helper-dependent replication-competent virus at a low frequency (Murakami et al., 2002). The generated atypical RCA was termed "helper-dependent E1-containing particle" (HDEP). As expected, the emergence of this type of particle was circumvented when a vector was used that lacked the sequence overlap with the E1 sequences in the genome of PER.C6.RTM. cells (Murakami et al., 2002). [0006] However, it now unexpectedly appears that using the system of matched vector and PER.C6.RTM. cells, some batches of recombinant adenovirus are contaminated at a very low frequency with particles that can cause cytopathological effect (CPE) in cells lacking E1 sequences. The particle is helper-dependent and contains E1-sequences and, therefore, is also an HDEP. This HDEP is generated in the absence of substantial homology between vector and packaging cell (see Murakami et al., 2004). [0007] While RCA is recognized by the regulatory authorities as a potential problem and detection of RCA in batches for clinical use is mandatory (USDHHS, FDA, CBER. Guidance for Industry, Guidance for human somatic cell therapy and gene therapy, March 1998), the safety aspects of HDEP are unclear. HDEP is replication deficient since it lacks the necessary viral genes for autonomous replication and, therefore, HDEP will not disseminate in a host. The theoretical possibility that the presence of a recombinant vector or wild-type adenovirus in the same cell may cause rescue and spread of HDEP cannot, however, be completely excluded. Hence, at least a latent need exists for means and methods for preventing generation of E1-containing particles during the preparation of batches of recombinant adenovirus particles. To this purpose, the present invention provides new cell lines, methods for preparing new cell lines, and the use thereof to make batches of RCA-free and HDEP-free recombinant adenovirus vectors. SUMMARY OF THE INVENTION [0008] Described herein are methods of generating a packaging cell comprising adenoviral E1A and E1B sequences, wherein E1A and at least one of the E1B coding regions are separated from each other in the genome of the cell. To this purpose, at least three embodiments are provided. [0009] In a first embodiment, E1A and E1B can be introduced into a precursor cell at different moments in time, reducing the chance that E1A and E1B are co-integrated on the same chromosomal location. [0010] In a second embodiment, the chances of co-integration into the same locus are reduced by introducing E1A and E1B coding sequences into a precursor cell on two different molecules that lack overlapping sequences that could otherwise lead to homologous recombination between these sequences. [0011] In a third embodiment, E1A and at least one of the E1B coding regions are introduced by virtue of one nucleic acid molecule whereon these sequences are separated by a given distance. It will be clear that in the third embodiment, instead of a single nucleic acid molecule, two or more nucleic acid molecules can also be introduced, which would satisfy the criterion that the E1A and at least one of the E1B coding regions are separated by a given distance, in case these two or more molecules would form one molecule, e.g., by ligation, recombination, and the like. The resulting packaging cells are characterized in that E1A and both E1B coding regions are present at a distance from each other and, hence, the chances are reduced of E1A and both E1B coding sequences together being incorporated into the genome of a recombinant adenovirus that is propagated on such packaging cells. [0012] The invention provides a method of preparing a cell capable of complementing E1-deficient adenoviral vectors without generating helper-dependent E1-containing particles, comprising the steps of: a) introducing into a precursor or ancestor of the cell nucleic acid sequence(s) coding for E1A gene functions and E1B gene functions, or if the precursor cell already comprises one of E1A or E1B gene functions, the other of the E1A and E1B gene functions; and b) selecting or identifying cells having obtained E1A and E1B in a chromosomal configuration that prevents the formation of helper-dependent E1-containing particles when the cells are used to complement a recombinant adenoviral vector deficient for one or more E1 gene functions, the vector lacking nucleic acid sequences that have substantial overlap with the chromosomally located E1 sequence that otherwise could give rise to homologous recombination. [0013] In one aspect, the invention provides a method of preparing a cell having adenovirus E1A and E1B-19K and E1B-55K coding sequences in its genome, wherein nucleic acid sequences comprising those sequences are introduced into a precursor cell, the method characterized in that: a) at least two nucleic acid molecules together comprising those sequences are introduced into the precursor cell, wherein E1A and at least one of the E1B coding sequences are present on different nucleic acid molecules, which are introduced into the precursor cell on a different moment in time, and wherein the precursor cell is not a baby rat kidney cell; or b) a nucleic acid molecule comprising the E1A coding sequence and a nucleic acid molecule comprising the E1B coding sequences are introduced into the precursor cell, wherein the nucleic acid molecules lack substantial overlap that could otherwise lead to homologous recombination between the at least two molecules; or c) the nucleic acid sequences comprising E1A and at least one of the E1B coding sequences are separated by at least 4 kb on a single nucleic acid molecule or on two or more nucleic acid molecules when these would form a single nucleic acid molecule. Preferably, in embodiment c), the sequences are separated by at least 10 kb, more preferably at least 34.5 kb or more. Preferably, the separating sequences are non-E1 sequences so that integration of both expression cassettes of E1A and E1B together in a single virus genome is rendered very unlikely or impossible. Alternatively, when the E1A and at least one of the E1B sequences are separated by 0 to 15 kb, a screen for the absence of inverted repeats of the E1-sequences in the generated cells may be used to select for cells, wherein the possibility of generating HDEP upon propagation of recombinant adenovirus in the generated cells is decreased or absent. The invention also provides cells obtainable by the method according to the invention. The invention further provides a cell comprising adenovirus E1A and E1B-55K and E1B-19K coding sequences in its genome, characterized in that the cell lacks stretches of nucleic acid sequence, wherein E1A and both E1B coding sequences are separated by less than 4 kb in the genome. Preferably, the cell lacks stretches of nucleic acid sequence, wherein E1A and both E1B coding sequences are separated by less than 10 kb in the genome, more preferably, the cell lacks stretches of nucleic acid sequence, wherein E1A and both E1B coding sequences are separated by less than 34.5 kb in the genome. [0014] It is another aspect of the invention to provide a method for providing or generating a cell comprising adenoviral E1 sequences, wherein E1 sequences include E1A and E1B coding sequences, characterized in that the method includes a step of selecting cells lacking inverted repeats comprising E1 sequences. The invention further provides a cell comprising adenovirus E1 sequences in its genome, wherein the E1 sequences include E1A and E1B coding sequences, characterized in that the E1 sequences are not present in the form of inverted repeats in the genome. In one embodiment thereof, the cell comprises at least two copies of the adenovirus sequences in its genome, which copies may be on the same chromosome. In another embodiment thereof, the cell comprises one copy of the E1 sequences in its genome, and further lacks sequences in its genome encoding (functional) pIX of an adenovirus. [0015] It is another aspect of the invention to provide a method for generating a batch of recombinant adenovirus having a deletion in the E1 region, comprising the steps of: a) introducing the recombinant adenovirus into a cell comprising E1 sequences of an adenovirus capable of complementing the deleted E1 sequences of the recombinant adenovirus; b) culturing the cell and harvesting the recombinant adenovirus, the method characterized in that the cell is a cell according to the invention. It will be clear to the person skilled in the art that instead of introducing the recombinant adenovirus, it is also possible to start the generation of recombinant adenovirus by using one or more nucleic acid fragments capable of forming the genome of the recombinant adenovirus and which will, after replication and packaging in that cell, form the recombinant adenovirus. Hence, for this embodiment, a recombinant adenovirus may also be a genome of the recombinant adenovirus. In a preferred embodiment, the recombinant adenovirus lacks substantial sequence overlap with the E1 sequences present in the cell, which could otherwise lead to homologous recombination. In yet another embodiment, the invention provides a packaging system comprising a packaging cell comprising E1 sequences in its genome and a recombinant adenovirus vector with a deletion in the E1 region, wherein the genome of the vector lacks substantial overlap with the E1 sequences in the genome of the packaging cell, characterized in that the packaging cell is a cell according to the present invention. BRIEF DESCRIPTION OF THE FIGURES [0016] FIG. 1 is a map of pIG.E1A. [0017] FIG. 2 depicts a map of pIG.E1AB21. [0018] FIG. 3 is an illustration of a map of pE1B. [0019] FIG. 4 depicts a map of pCC.E1A. Continue reading about Packaging cells for recombinant adenovirus... 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